The hydroxyphenylpyruvate dioxygenases are enzymes which catalyse the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. This reaction takes place in the presence of iron (Fe2+) and in the presence of oxygen (Crouch, N. P. et al., Tetrahedron, 53, 20, 6993-7010, 1997). It may be hypothesized that the HPPDs contain an active site which is capable of catalysing this reaction, in which iron, the substrate and the molecule of oxygen link together, although such an active site has not so far been described.
Some molecules which inhibit this enzyme, and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate, are also known. Some of these molecules have been used as herbicides since inhibition of the reaction in plants leads to whitening of the leaves of the treated plants and to the death of the said plants (Pallett, K. E. et al. 1997 Pestic. Sci. 50 83-84). The herbicides for which HPPD is the target, and which are described in the state of the art, are, in particular, isoxazoles (EP418175, EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, which is a selective herbicide for maize, diketonitriles (EP496630, EP496631), in particular 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2phenyl)propane-1,3-dione, triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particular sulcotrione, or else pyrazolinates.
Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. etal., J.Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor.
It is this third strategy which was described for successfully obtaining plants which were tolerant to HPPD inhibitors (WO96/38567), with it being understood that this was the first time that a strategy of simply overexpressing the (non-mutated) sensitive target enzyme was successfully used for conferring on plants agricultural level tolerance to a herbicide.
Despite the success obtained with this strategy of simply overexpressing the target enzyme, it is still necessary to improve the system of tolerance to HPPD inhibitors in order to obtain a tolerance whatever the conditions under which the tolerant plants are cultivated, or the commercial doses at which the herbicides are applied in the fields, may be.